Sensory satellite glial Gq-GPCR activation alleviates inflammatory pain via peripheral adenosine 1 receptor activation.

Glial fibrillary acidic protein expressing (GFAP+) glia modulate nociceptive neuronal activity in both the peripheral nervous system (PNS) and the central nervous system (CNS). Resident GFAP+ glia in dorsal root ganglia (DRG) known as satellite glial cells (SGCs) potentiate neuronal activity by releasing pro-inflammatory cytokines and neuroactive compounds. In this study, we tested the hypothesis that SGC Gq-coupled receptor (Gq-GPCR) signaling modulates pain sensitivity in vivo using Gfap-hM3Dq mice. Complete Freund's adjuvant (CFA) was used to induce inflammatory pain, and mechanical sensitivity and thermal sensitivity were used to assess the neuromodulatory effect of glial Gq-GPCR activation in awake mice. Pharmacogenetic activation of Gq-GPCR signaling in sensory SGCs decreased heat-induced nociceptive responses and reversed inflammation-induced mechanical allodynia via peripheral adenosine A1 receptor activation. These data reveal a previously unexplored role of sensory SGCs in decreasing afferent excitability. The identified molecular mechanism underlying the analgesic role of SGCs offers new approaches for reversing peripheral nociceptive sensitization.

Multiple Lines of Evidence Indicate That Gliotransmission Does Not Occur under Physiological Conditions.

A major controversy persists within the field of glial biology concerning whether or not, under physiological conditions, neuronal activity leads to Ca2+-dependent release of neurotransmitters from astrocytes, a phenomenon known as gliotransmission. Our perspective is that, while we and others can apply techniques to cause gliotransmission, there is considerable evidence gathered using astrocyte-specific and more physiological approaches which suggests that gliotransmission is a pharmacological phenomenon rather than a physiological process. Approaches providing evidence against gliotransmission include stimulation of Gq-GPCRs expressed only in astrocytes, as well as removal of the primary proposed source of astrocyte Ca2+ responsible for gliotransmission. These approaches contrast with those supportive of gliotransmission, which include mechanical stimulation, strong astrocytic depolarization using whole-cell patch-clamp or optogenetics, uncaging Ca2+ or IP3, chelating Ca2+ using BAPTA, and nonspecific bath application of agonists to receptors expressed by a multitude of cell types. These techniques are not subtle and therefore are not supportive of recent suggestions that gliotransmission requires very specific and delicate temporal and spatial requirements. Other evidence, including lack of propagating Ca2+ waves between astrocytes in healthy tissue, lack of expression of vesicular release machinery, and the demise of the d-serine gliotransmission hypothesis, provides additional evidence against gliotransmission. Overall, the data suggest that Ca2+-dependent release of neurotransmitters is the province of neurons, not astrocytes, in the intact brain under physiological conditions.Dual Perspectives Companion Paper: Gliotransmission: Beyond Black-and-White, by Iaroslav Savtchouk and Andrea Volterra.

Ganglionic GFAP + glial Gq-GPCR signaling enhances heart functions in vivo.

The sympathetic nervous system (SNS) accelerates heart rate, increases cardiac contractility, and constricts resistance vessels. The activity of SNS efferent nerves is generated by a complex neural network containing neurons and glia. Gq G protein-coupled receptor (Gq-GPCR) signaling in glial fibrillary acidic protein-expressing (GFAP+) glia in the central nervous system supports neuronal function and regulates neuronal activity. It is unclear how Gq-GPCR signaling in GFAP+ glia affects the activity of sympathetic neurons or contributes to SNS-regulated cardiovascular functions. In this study, we investigated whether Gq-GPCR activation in GFAP+ glia modulates the regulatory effect of the SNS on the heart; transgenic mice expressing Gq-coupled DREADD (designer receptors exclusively activated by designer drugs) (hM3Dq) selectively in GFAP+ glia were used to address this question in vivo. We found that acute Gq-GPCR activation in peripheral GFAP+ glia significantly accelerated heart rate and increased left ventricle contraction. Pharmacological experiments suggest that the glial-induced cardiac changes were due to Gq-GPCR activation in satellite glial cells within the sympathetic ganglion; this activation led to increased norepinephrine (NE) release and beta-1 adrenergic receptor activation within the heart. Chronic glial Gq-GPCR activation led to hypotension in female Gfap-hM3Dq mice. This study provides direct evidence that Gq-GPCR activation in peripheral GFAP+ glia regulates cardiovascular functions in vivo.

Molecular approaches for manipulating astrocytic signaling in vivo.

Astrocytes are the predominant glial type in the central nervous system and play important roles in assisting neuronal function and network activity. Astrocytes exhibit complex signaling systems that are essential for their normal function and the homeostasis of the neural network. Altered signaling in astrocytes is closely associated with neurological and psychiatric diseases, suggesting tremendous therapeutic potential of these cells. To further understand astrocyte function in health and disease, it is important to study astrocytic signaling in vivo. In this review, we discuss molecular tools that enable the selective manipulation of astrocytic signaling, including the tools to selectively activate and inactivate astrocyte signaling in vivo. Lastly, we highlight a few tools in development that present strong potential for advancing our understanding of the role of astrocytes in physiology, behavior, and pathology.

Astrocyte calcium signaling: from observations to functions and the challenges therein.

We provide an overview of recent progress on the study of astrocyte intracellular Ca(2+) signaling. We consider the methods that have been used to monitor astrocyte Ca(2+) signals, the various types of Ca(2+) signals that have been discovered (waves, microdomains, and intrinsic fluctuations), the approaches used to broadly trigger and block Ca(2+) signals, and, where possible, the proposed and demonstrated physiological roles for astrocyte Ca(2+) signals within neuronal microcircuits. Although important progress has been made, we suggest that further detailed work is needed to explore the biophysics and molecular mechanisms of Ca(2+) signaling within entire astrocytes, including their fine distal extensions, such as processes that interact spatially with neurons and blood vessels. Improved methods are also needed to mimic and block molecularly defined types of Ca(2+) signals within genetically specified populations of astrocytes. Moreover, it will be essential to study astrocyte Ca(2+) activity in vivo to distinguish between pharmacological and physiological activity, and to study Ca(2+) activity in situ to rigorously explore mechanisms. Once methods to reliably measure, mimic, and block specific astrocyte Ca(2+) signals with high temporal and spatial precision are available, researchers will be able to carefully explore the correlative and causative roles that Ca(2+) signals may play in the functions of astrocytes, blood vessels, neurons, and microcircuits in the healthy and diseased brain.

Astrocyte IP3R2-dependent Ca(2+) signaling is not a major modulator of neuronal pathways governing behavior.

Calcium-dependent release of gliotransmitters by astrocytes is reported to play a critical role in synaptic transmission and be necessary for long-term potentiation (LTP), long-term depression (LTD) and other forms of synaptic modulation that are correlates of learning and memory. Further, physiological processes reported to be dependent on Ca(2+) fluxes in astrocytes include functional hyperemia, sleep, and regulation of breathing. The preponderance of findings indicate that most, if not all, receptor dependent Ca(2+) fluxes within astrocytes are due to release of Ca(2+) through IP3 receptor/channels in the endoplasmic reticulum. Findings from several laboratories indicate that astrocytes only express IP3 receptor type 2 (IP3R2) and that a knockout of IP3R2 obliterates the GPCR-dependent astrocytic Ca(2+) responses. Assuming that astrocytic Ca(2+) fluxes play a critical role in synaptic physiology, it would be predicted that elimination of astrocytic Ca(2+) fluxes would lead to marked changes in behavioral tests. Here, we tested this hypothesis by conducting a broad series of behavioral tests that recruited multiple brain regions, on an IP3R2 conditional knockout mouse model. We present the novel finding that behavioral processes are unaffected by lack of astrocyte IP3R-mediated Ca(2+) signals. IP3R2 cKO animals display no change in anxiety or depressive behaviors, and no alteration to motor and sensory function. Morris water maze testing, a behavioral correlate of learning and memory, was unaffected by lack of astrocyte IP3R2-mediated Ca(2+)-signaling. Therefore, in contrast to the prevailing literature, we find that neither receptor-driven astrocyte Ca(2+) fluxes nor, by extension, gliotransmission is likely to be a major modulating force on the physiological processes underlying behavior.

Astrocytic Gq-GPCR-linked IP3R-dependent Ca2+ signaling does not mediate neurovascular coupling in mouse visual cortex in vivo.

Local blood flow is modulated in response to changing patterns of neuronal activity (Roy and Sherrington, 1890), a process termed neurovascular coupling. It has been proposed that the central cellular pathway driving this process is astrocytic Gq-GPCR-linked IP3R-dependent Ca(2+) signaling, though in vivo tests of this hypothesis are largely lacking. We examined the impact of astrocytic Gq-GPCR and IP3R-dependent Ca(2+) signaling on cortical blood flow in awake, lightly sedated, responsive mice using multiphoton laser-scanning microscopy and novel genetic tools that enable the selective manipulation of astrocytic signaling pathways in vivo. Selective stimulation of astrocytic Gq-GPCR cascades and downstream Ca(2+) signaling with the hM3Dq DREADD (designer receptors exclusively activated by designer drugs) designer receptor system was insufficient to modulate basal cortical blood flow. We found no evidence of observable astrocyte endfeet Ca(2+) elevations following physiological visual stimulation despite robust dilations of adjacent arterioles using cyto-GCaMP3 and Lck-GCaMP6s, the most sensitive Ca(2+) indicator available. Astrocytic Ca(2+) elevations could be evoked when inducing the startle response with unexpected air puffs. However, startle-induced astrocytic Ca(2+) signals did not precede corresponding startle-induced hemodynamic changes. Further, neurovascular coupling was intact in lightly sedated, responsive mice genetically lacking astrocytic IP3R-dependent Ca(2+) signaling (IP3R2 KO). These data demonstrate that astrocytic Gq-GPCR-linked IP3R-dependent Ca(2+) signaling does not mediate neurovascular coupling in visual cortex of awake, lightly sedated, responsive mice.

Evolutionary etiology of high-grade astrocytomas.

Glioblastoma (GBM), the most common brain malignancy, remains fatal with no effective treatment. Analyses of common aberrations in GBM suggest major regulatory pathways associated with disease etiology. However, 90% of GBMs are diagnosed at an advanced stage (primary GBMs), providing no access to early disease stages for assessing disease progression events. As such, both understanding of disease mechanisms and the development of biomarkers and therapeutics for effective disease management are limited. Here, we describe an adult-inducible astrocyte-specific system in genetically engineered mice that queries causation in disease evolution of regulatory networks perturbed in human GBM. Events yielding disease, both engineered and spontaneous, indicate ordered grade-specific perturbations that yield high-grade astrocytomas (anaplastic astrocytomas and GBMs). Impaired retinoblastoma protein RB tumor suppression yields grade II histopathology. Additional activation of v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) network drives progression to grade III disease, and further inactivation of phosphatase and tensin homolog (PTEN) yields GBM. Spontaneous missense mutation of tumor suppressor Trp53 arises subsequent to KRAS activation, but before grade III progression. The stochastic appearance of mutations identical to those observed in humans, particularly the same spectrum of p53 amino acid changes, supports the validity of engineered lesions and the ensuing interpretations of etiology. Absence of isocitrate dehydrogenase 1 (IDH1) mutation, asymptomatic low grade disease, and rapid emergence of GBM combined with a mesenchymal transcriptome signature reflect characteristics of primary GBM and provide insight into causal relationships.

Modulation of the autonomic nervous system and behaviour by acute glial cell Gq protein-coupled receptor activation in vivo.

Glial fibrillary acidic protein (GFAP)-expressing cells (GFAP(+) glial cells) are the predominant cell type in the central and peripheral nervous systems. Our understanding of the role of GFAP(+) glial cells and their signalling systems in vivo is limited due to our inability to manipulate these cells and their receptors in a cell type-specific and non-invasive manner. To circumvent this limitation, we developed a transgenic mouse line (GFAP-hM3Dq mice) that expresses an engineered Gq protein-coupled receptor (Gq-GPCR) known as hM3Dq DREADD (designer receptor exclusively activated by designer drug) selectively in GFAP(+) glial cells. The hM3Dq receptor is activated solely by a pharmacologically inert, but bioavailable, ligand (clozapine-N-oxide; CNO), while being non-responsive to endogenous GPCR ligands. In GFAP-hM3Dq mice, CNO administration increased heart rate, blood pressure and saliva formation, as well as decreased body temperature, parameters that are controlled by the autonomic nervous system (ANS). Additionally, changes in activity-related behaviour and motor coordination were observed following CNO administration. Genetically blocking inositol 1,4,5-trisphosphate (IP3)-dependent Ca(2+) increases in astrocytes failed to interfere with CNO-mediated changes in ANS function, locomotor activity or motor coordination. Our findings reveal an unexpectedly broad role of GFAP(+) glial cells in modulating complex physiology and behaviour in vivo and suggest that these effects are not dependent on IP3-dependent increases in astrocytic Ca(2+).

Morphological and behavioral changes in the pathogenesis of a novel mouse model of communicating hydrocephalus.

The Ro1 model of hydrocephalus represents an excellent model for studying the pathogenesis of hydrocephalus due to its complete penetrance and inducibility, enabling the investigation of the earliest cellular and histological changes in hydrocephalus prior to overt pathology. Hematoxylin and eosin staining, immunofluorescence and electron microscopy were used to characterize the histopathological events of hydrocephalus in this model. Additionally, a broad battery of behavioral tests was used to investigate behavioral changes in the Ro1 model of hydrocephalus. The earliest histological changes observed in this model were ventriculomegaly and disorganization of the ependymal lining of the aqueduct of Sylvius, which occurred concomitantly. Ventriculomegaly led to thinning of the ependyma, which was associated with periventricular edema and areas of the ventricular wall void of cilia and microvilli. Ependymal denudation was subsequent to severe ventriculomegaly, suggesting that it is an effect, rather than a cause, of hydrocephalus in the Ro1 model. Additionally, there was no closure of the aqueduct of Sylvius or any blockages within the ventricular system, even with severe ventriculomegaly, suggesting that the Ro1 model represents a model of communicating hydrocephalus. Interestingly, even with severe ventriculomegaly, there were no behavioral changes, suggesting that the brain is able to compensate for the structural changes that occur in the pathogenesis of hydrocephalus if the disorder progresses at a sufficiently slow rate.

Implication of Kir4.1 channel in excess potassium clearance: an in vivo study on anesthetized glial-conditional Kir4.1 knock-out mice.

The K(ir)4.1 channel is crucial for the maintenance of the resting membrane potential of glial cells, and it is believed to play a main role in the homeostasis of extracellular potassium. To understand its importance in these two phenomena, we have measured in vivo the variations of extracellular potassium concentration ([K(+)](o)) (with potassium-sensitive microelectrodes) and membrane potential of glial cells (with sharp electrodes) during stimulations in wild-type (WT) mice and glial-conditional knock-out (cKO) K(ir)4.1 mice. The conditional knockout was driven by the human glial fibrillary acidic protein promoter, gfa2. Experiments were performed in the hippocampus of anesthetized mice (postnatal days 17-24). Low level stimulation (<20 stimuli, 10 Hz) induced a moderated increase of [K(+)](o) (<2 mm increase) in both WT and cKO mice. However, cKO mice exhibited slower recovery of [K(+)](o) levels. With long-lasting stimulation (300 stimuli, 10 Hz), [K(+)](o) in WT and cKO mice displayed characteristic ceiling level (>2 mm increase) and recovery undershoot, with a more pronounced and prolonged undershoot in cKO mice. In addition, cKO glial cells were more depolarized, and, in contrast to those from WT mice, their membrane potential did not follow the stimulation-induced [K(+)](o) changes, reflecting the loss of their high potassium permeability. Our in vivo results support the role of K(ir)4.1 in setting the membrane potential of glial cells and its contribution to the glial potassium permeability. In addition, our data confirm the necessity of the K(ir)4.1 channel for an efficient uptake of K(+) by glial cells.

Hippocampal short- and long-term plasticity are not modulated by astrocyte Ca2+ signaling.

The concept that astrocytes release neuroactive molecules (gliotransmitters) to affect synaptic transmission has been a paradigm shift in neuroscience research over the past decade. This concept suggests that astrocytes, together with pre- and postsynaptic neuronal elements, make up a functional synapse. Astrocyte release of gliotransmitters (for example, glutamate and adenosine triphosphate) is generally accepted to be a Ca2+-dependent process. We used two mouse lines to either selectively increase or obliterate astrocytic Gq G protein-coupled receptor Ca2+ signaling to further test the hypothesis that astrocytes release gliotransmitters in a Ca2+-dependent manner to affect synaptic transmission. Neither increasing nor obliterating astrocytic Ca2+ fluxes affects spontaneous and evoked excitatory synaptic transmission or synaptic plasticity. Our findings suggest that, at least in the hippocampus, the mechanisms of gliotransmission need to be reconsidered.

Sorting out astrocyte physiology from pharmacology.

A number of exciting findings have been made in astrocytes during the past 15 years that have led many researchers to redefine how the brain works. Astrocytes are now widely regarded as cells that propagate Ca(2+) over long distances in response to stimulation, and, similar to neurons, release transmitters (called gliotransmitters) in a Ca(2+)-dependent manner to modulate a host of important brain functions. Although these discoveries have been very exciting, it is essential to place them in the proper context of the approaches used to obtain them to determine their relevance to brain physiology. This review revisits the key observations made in astrocytes that greatly impact how they are thought to regulate brain function, including the existence of widespread propagating intercellular Ca(2+) waves, data suggesting that astrocytes signal to neurons through Ca(2+)-dependent release of glutamate, and evidence for the presence of vesicular machinery for the regulated exocytosis of gliotransmitters.

What is the role of astrocyte calcium in neurophysiology?

Astrocytes comprise approximately half of the volume of the adult mammalian brain and are the primary neuronal structural and trophic supportive elements. Astrocytes are organized into distinct nonoverlapping domains and extend elaborate and dense fine processes that interact intimately with synapses and cerebrovasculature. The recognition in the mid 1990s that astrocytes undergo elevations in intracellular calcium concentration following activation of G protein-coupled receptors by synaptically released neurotransmitters demonstrated not only that astrocytes display a form of excitability but also that astrocytes may be active participants in brain information processing. The roles that astrocytic calcium elevations play in neurophysiology and especially in modulation of neuronal activity have been intensely researched in recent years. This review will summarize the current understanding of the function of astrocytic calcium signaling in neurophysiological processes and discuss areas where the role of astrocytes remains controversial and will therefore benefit from further study.

Engineering GPCR signaling pathways with RASSLs.

We are creating families of designer G protein-coupled receptors (GPCRs) to allow for precise spatiotemporal control of GPCR signaling in vivo. These engineered GPCRs, called receptors activated solely by synthetic ligands (RASSLs), are unresponsive to endogenous ligands but can be activated by nanomolar concentrations of pharmacologically inert, drug-like small molecules. Currently, RASSLs exist for the three major GPCR signaling pathways (G(s), G(i) and G(q)). We review these advances here to facilitate the use of these powerful and diverse tools.

Loss of IP3 receptor-dependent Ca2+ increases in hippocampal astrocytes does not affect baseline CA1 pyramidal neuron synaptic activity.

Astrocytes in the hippocampus release calcium (Ca(2+)) from intracellular stores intrinsically and in response to activation of G(q)-linked G-protein-coupled receptors (GPCRs) through the binding of inositol 1,4,5-trisphosphate (IP(3)) to its receptor (IP(3)R). Astrocyte Ca(2+) has been deemed necessary and sufficient to trigger the release of gliotransmitters, such as ATP and glutamate, from astrocytes to modulate neuronal activity. Several lines of evidence suggest that IP(3)R type 2 (IP(3)R2) is the primary IP(3)R expressed by astrocytes. To determine whether IP(3)R2 is the primary functional IP(3)R responsible for astrocytic Ca(2+) increases, we conducted experiments using an IP(3)R2 knock-out mouse model (IP(3)R2 KO). We show, for the first time, that lack of IP(3)R2 blocks both spontaneous and G(q)-linked GPCR-mediated increases in astrocyte Ca(2+). Furthermore, neuronal G(q)-linked GPCR Ca(2+) increases remain intact, suggesting that IP(3)R2 does not play a major functional role in neuronal calcium store release or may not be expressed in neurons. Additionally, we show that lack of IP(3)R2 in the hippocampus does not affect baseline excitatory neuronal synaptic activity as measured by spontaneous EPSC recordings from CA1 pyramidal neurons. Whole-cell recordings of the tonic NMDA receptor-mediated current indicates that ambient glutamate levels are also unaffected in the IP(3)R2 KO. These data show that IP(3)R2 is the key functional IP(3)R driving G(q)-linked GPCR-mediated Ca(2+) increases in hippocampal astrocytes and that removal of astrocyte Ca(2+) increases does not significantly affect excitatory neuronal synaptic activity or ambient glutamate levels.

Reinduction of ErbB2 in astrocytes promotes radial glial progenitor identity in adult cerebral cortex.

Radial glial cells play a critical role in the construction of mammalian brain by functioning as a source of new neurons and by providing a scaffold for radial migration of new neurons to their target locations. Radial glia transform into astrocytes at the end of embryonic development. Strategies to promote functional recovery in the injured adult brain depend on the generation of new neurons and the appropriate guidance of these neurons to where they are needed, two critical functions of radial glia. Thus, the competence to regain radial glial identity in the adult brain is of significance for the ability to promote functional repair via neurogenesis and targeted neuronal migration in the mature brain. Here we show that the in vivo induction of the tyrosine kinase receptor, ErbB2, in mature astrocytes enables a subset of them to regain radial glial identity in the mature cerebral cortex. These new radial glial progenitors are capable of giving rise to new neurons and can support neuronal migration. These studies indicate that ErbB2 signaling critically modulates the functional state of radial glia, and induction of ErbB2 in distinct adult astrocytes can promote radial glial identity in the mature cerebral cortex.

Conditional knock-out of Kir4.1 leads to glial membrane depolarization, inhibition of potassium and glutamate uptake, and enhanced short-term synaptic potentiation.

During neuronal activity, extracellular potassium concentration ([K+]out) becomes elevated and, if uncorrected, causes neuronal depolarization, hyperexcitability, and seizures. Clearance of K+ from the extracellular space, termed K+ spatial buffering, is considered to be an important function of astrocytes. Results from a number of studies suggest that maintenance of [K+]out by astrocytes is mediated by K+ uptake through the inward-rectifying Kir4.1 channels. To study the role of this channel in astrocyte physiology and neuronal excitability, we generated a conditional knock-out (cKO) of Kir4.1 directed to astrocytes via the human glial fibrillary acidic protein promoter gfa2. Kir4.1 cKO mice die prematurely and display severe ataxia and stress-induced seizures. Electrophysiological recordings revealed severe depolarization of both passive astrocytes and complex glia in Kir4.1 cKO hippocampal slices. Complex cell depolarization appears to be a direct consequence of Kir4.1 removal, whereas passive astrocyte depolarization seems to arise from an indirect developmental process. Furthermore, we observed a significant loss of complex glia, suggestive of a role for Kir4.1 in astrocyte development. Kir4.1 cKO passive astrocytes displayed a marked impairment of both K+ and glutamate uptake. Surprisingly, membrane and action potential properties of CA1 pyramidal neurons, as well as basal synaptic transmission in the CA1 stratum radiatum appeared unaffected, whereas spontaneous neuronal activity was reduced in the Kir4.1 cKO. However, high-frequency stimulation revealed greatly elevated posttetanic potentiation and short-term potentiation in Kir4.1 cKO hippocampus. Our findings implicate a role for glial Kir4.1 channel subunit in the modulation of synaptic strength.

Selective stimulation of astrocyte calcium in situ does not affect neuronal excitatory synaptic activity.

Astrocytes are considered the third component of the synapse, responding to neurotransmitter release from synaptic terminals and releasing gliotransmitters--including glutamate--in a Ca(2+)-dependent manner to affect neuronal synaptic activity. Many studies reporting astrocyte-driven neuronal activity have evoked astrocyte Ca(2+) increases by application of endogenous ligands that directly activate neuronal receptors, making astrocyte contribution to neuronal effect(s) difficult to determine. We have made transgenic mice that express a Gq-coupled receptor only in astrocytes to evoke astrocyte Ca(2+) increases using an agonist that does not bind endogenous receptors in brain. By recording from CA1 pyramidal cells in acute hippocampal slices from these mice, we demonstrate that widespread Ca(2+) elevations in 80%-90% of stratum radiatum astrocytes do not increase neuronal Ca(2+), produce neuronal slow inward currents, or affect excitatory synaptic activity. Our findings call into question the developing consensus that Ca(2+)-dependent glutamate release by astrocytes directly affects neuronal synaptic activity in situ.